VEGFA locus amplification potentially predicts a favorable prognosis in gastric adenocarcinoma.

Gastric adenocarcinoma harbors a range of genetic and epigenetic alterations, including alterations in DNA copy number. However, the key genes that promote the development and progression of gastric adenocarcinoma remain unknown. To identify the key genes amplified in gastric adenocarcinoma, we performed array comparative genomic hybridization on formalin-fixed paraffin-embedded samples of surgically resected gastric adenocarcinoma. We detected a relatively wide genomic region of gain containing the vascular endothelial growth factor A (VEGFA) gene locus on chromosome 6p. VEGFA locus amplification in gastric adenocarcinoma was validated by fluorescence in situ hybridization. To assess the frequency of VEGFA locus amplification in gastric adenocarcinoma, we conducted multiplex ligation-dependent probe amplification (MLPA) assays using homemade probes designed to target the VEGFA gene locus. Eleven of 54 (20 %) gastric adenocarcinomas with MLPA values above 1.3 were defined as having VEGFA locus amplification. Next, we investigated the effect of VEGFA locus amplification on the clinicopathological characteristics of gastric adenocarcinomas and patient survival. VEGFA locus amplification demonstrated a significantly close relationship with pathological intestinal type and lower rates of venous invasion Furthermore, a Kaplan-Meier analysis showed that patients with VEGFA locus amplification had significantly better overall survival than those without amplification (p = 0.038), particularly in the long-term follow-up period. In conclusion, VEGFA locus amplification can predict modest aggressiveness and good outcomes, suggesting the possibility that it may predict a favorable prognosis in patients with gastric adenocarcinoma.

Suitability of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration versus Paired Transbronchial Biopsy Specimens for Evaluating Programmed Death Ligand-1 Expression in Stage III and IV Lung Cancer: A Comparative Retrospective Study.

Objectives: Cancer cells usually escape tumor-reactive T-cell responses using immune checkpoint proteins, such as programmed death protein-1 (PD-1) and its ligand, programmed death ligand-1 (PD-L1). These proteins can be blocked by immune checkpoint inhibitors (ICIs); the decision on ICI-based first-line treatment for advanced lung cancers depends on the PD-L1 levels in tumor specimens. Determining the PD-L1 expression conventionally requires histological specimens from resected tumors and core biopsy specimens. Non-small cell lung cancer (NSCLC) is usually diagnosed at stage III or IV; therefore, only small biopsy specimens, such as those obtained via endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) are available. However, the suitability of EBUS-TBNA specimens determining the PD-L1 expression levels in advanced lung cancers remains unclear. Materials and Methods: Here, we investigated the concordance rate of PD-L1 expression between EBUS-TBNA and matched transbronchial biopsy (TBB) specimens. Using the 22C3 anti-PD-L1 antibody (immunohistochemistry), we determined the PD-L1 expression levels in paired specimens obtained from 69 patients (50 with advanced NSCLC and 19 with small cell lung cancer [SCLC]), as well as the efficacy of ICIs in these patients. Results: The concordance rate of PD-L1 expression between the EBUS-TBNA and TBB specimens was 78.3%. The κ values referent to the PD-L1-positive expression rate between EBUS-TBNA and TBB specimens were 0.707 and 0.676 at cutoff limits of ≥1% and ≥50%, respectively. Among the 19 SCLC patients, 16 (84.2%) exhibited no PD-L1 expression in both EBUS-TBNA and TBB specimens. Notably, the progression-free survival of patients with ≥50% PD-L1 expression in the paired specimens who received ICI treatment was 8.3 months. Conclusion: Collectively, our results validate the use of EBUS-TBNA specimens for the determination of the PD-L1 expression levels in the context of NSCLC and SCLC.

Neural EGFL like 2 expressed in myoepithelial cells and suppressed breast cancer cell migration.

Breast tissue has a branching structure that contains double-layered cells, consisting primarily of luminal epithelial cells inside and myoepithelial cells outside. Ductal carcinoma in situ (DCIS) still has myoepithelial cells surrounding the cancer cells. However, myoepithelial cells disappear in invasive ductal carcinoma. In this study, we detected expression of neural EGFL like (NELL) 2 and one of its receptors, roundabout guidance receptor (ROBO) 3, in myoepithelial and luminal epithelial cells (respectively) in normal breast tissue. NELL2 also was expressed in myoepithelial cells surrounding the non-cancerous intraductal proliferative lesions and DCIS. However, the expression level and proportion of NELL2-positive cells in DCIS were lower than those in normal and non-cancerous intraductal proliferative lesions. ROBO3 expression was decreased in invasive ductal carcinoma compared to that in normal and non-cancerous intraductal proliferative lesions. An evaluation of NELL2's function in breast cancer cell lines demonstrated that full-length NELL2 suppressed cell adhesion and migration in vitro. In contrast, the N-terminal domain of NELL2 increased cell adhesion in the early phase and migration in vitro in some breast cancer cells. These results suggested that full-length NELL2 protein, when expressed in myoepithelial cells, might serve as an inhibitor of breast cancer cell migration.

Semi-comprehensive analysis of gene amplification in thymic malignant tumors using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

Research on the amplification of oncogenes in thymic malignant tumor is limited. In this study, we aimed to determine the gene amplification status of receptor tyrosine kinases and other cell regulator genes in thymic malignant tumors, with a view toward the future introduction of molecular targeted therapy. In addition, we examined the usefulness of multiplex, ligation-dependent probe amplification (MLPA) in the semi-comprehensive detection of these gene amplifications. The participants of this study were nine patients with thymic carcinoma and one patient with atypical carcinoid who underwent resection at our department from 1999 to 2016. Twenty-four oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, BRAF, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR) were analyzed for amplification by MLPA. In cases where amplification by MLPA was suspected, confirmation was performed by fluorescence in situ hybridization (FISH). Immunostaining for detected oncoproteins and p53 were performed in cases with confirmed oncogene amplification. MYC (2/10, 20%) and MDM2 (1/10, 10%) amplifications were detected using MLPA and FISH. Immunostaining in both cases was positive. The MDM2-amplified tumor relapsed and spread rapidly after operation despite the use of post-operative chemo-radiotherapy. MYC amplification may be involved in the carcinogenesis of thymic malignant tumors. In addition, MDM2 amplification may be a concern in the increased malignancy.

The relation between anti-TGBFR1 immunohistochemical reaction and low Ki67, small tumor size and high estrogen receptor expression in invasive breast cancer.

Most breast cancers are derived from the luminal epithelium, which composes the inside of the breast ductal structure. Ductal carcinoma in situ (DCIS) leads to invasive ductal carcinoma, but noncancerous intraductal proliferative lesions are also a risk factor for ductal carcinoma. The transforming growth factor beta (TGFB) signaling pathway behaves as a tumor suppressor in the early stage of cancer, and conversely as a tumor growth factor in invasive stages in several cancers. In this study, we performed immunohistochemistry with an antibody that detects the cytoplasmic region of TGFB receptor 1 (TGFBR1) and elucidated TGFBR1 protein expression in luminal epithelial cells of noncancerous breast ducts and in several cases of DCIS and invasive carcinoma. TGFBR1 expression was higher in noncancerous breast tissue than in cancerous tissue, and a difference in expression was also seen among histological subtypes. Comparing the expression level of TGFBR1 in cancer cells and clinico-pathological parameters, cases expressing low TGFBR1 tended to show low estrogen receptor expression, large tumor size (≥10 mm), and a high Ki67 labeling index. These data suggested that TGFBR1 protein expression may be related to the suppression of breast cancer cell growth.

Screening of the copy number increase of AKT in lung carcinoma by custom-designed MLPA.

Treatments for lung cancer include therapies targeting aberrant oncoproteins, but there remains a high medical need for novel therapies. Our previous studies showed that gene amplification/high-level polysomy of AKT1/2 occurs in more than 10% of lung carcinomas. Here, we describe multiplex ligation-dependent probe amplification analysis (MLPA) as a high-throughput method to evaluate copy number increases (CNIs) of AKT1/2 in lung carcinomas. The performance of MLPA using custom-made probes in formalin-fixed paraffin-embedded tissue was evaluated by comparing it to immunohistochemistry and fluorescence in situ hybridization analysis (FISH). By MLPA, we found 4 out of 30 samples harboring gene "gain" when the conventional cutoff value (> 1.3) was used. Two samples with gene amplification by FISH had MLPA values of 1.85 and 1.75, which were lower than the conventional cutoff for "amplification" (> 2.0). Moreover, samples with CNIs due to polysomy by FISH gave MLPA values between 1.13 and 1.47, so some samples had lower values than 1.3. The reasons appeared to be stromal contamination and the presence of carcinoma cells without CNIs. However, when we changed the cutoff for "gain" to the "average+2xstandard error", we detected CNIs in 10 samples, with only one each of false-positive and false-negative results. The sensitivity was 90% and the specificity was 98%. Consistently, all cases exhibiting CNI by this criteria revealed Akt activation. In conclusion, MLPA implemented with custom-made probes and an optimized cutoff value is a feasible screening method to semi-quantitatively detect oncogene aberrations, and may contribute to the design of individualized, molecularly targeted therapies against lung carcinoma.

Amplicons in breast cancers analyzed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

Gene amplification is a common event in breast cancer, and identifies actual and potential targets of molecular therapy. The aim of the present study was to determine the amplification status of 22 genes that are reportedly frequently amplified in breast cancers. An archive of 322 formalin-fixed and paraffin-embedded invasive breast cancer tissues were screened by multiple ligation-dependent probe amplification (MLPA) and a total of 906 gene loci judged as 'gain' or 'amplified' was further confirmed to have been amplified based on fluorescence in situ hybridization (FISH). The results showed that 109 of 322 tumors (34%) displayed gene amplification of at least one of the 22 genes. The frequencies of the amplification of four regions containing known driver oncogenes were as follows: 8p11 (ZNF703, FGFR1, ADAM9, IKBKB), 8q24 (MYC), 11q13 (CCND1, C11ORF30), and 17q11-21 (CPD, MED1, ERBB2, CDC6, TOP2A, MAPT) exhibited amplification in 9.6%, 9.6%, 12.4%, and 12.1% of the tumors, respectively. In addition to homogeneously staining region- or double-minute chromosome-type amplifications, a centromere-associated-type amplification was found in nine tumors. Co-localization of the amplicon on 8p11 and the amplicon on 11q13 in single cells was found in 10 tumors, and in six of those tumors the two amplicons constituted single amplification units. Similarly, an amplicon consisting of ERBB2 and its flanking genes on 17q12-21 co-localized with an amplicon on 8p11 in 10 tumors and with the amplicon on 11q13 in five tumors. Thus, precise and feasible analysis of gene amplification status can be obtained using a combination of MLPA and FISH.

A novel combination of bortezomib, lenalidomide, and clarithromycin produced stringent complete response in refractory multiple myeloma complicated with diabetes mellitus - clinical significance and possible mechanisms: a case report.

BACKGROUND: In general, dexamethasone is a required component drug in various combination chemotherapies for treating multiple myeloma, and its efficacy has been widely recognized. However, administration of dexamethasone is known to cause various adverse effects including hyperglycemia which requires insulin therapy. During the course of treatment, we developed a novel effective dexamethasone-free combination regimen and evaluated it for its effect in multiple myeloma. CASE PRESENTATION: We report a case of 68-year-old Japanese woman with refractory advanced Bence-Jones-λ type multiple myeloma associated with diabetes mellitus. Various combination regimens were carried out, but the response to some regimens was insufficient or others containing dexamethasone, although effective, were inappropriate to continue due to aggravation of diabetes mellitus. Thus, we developed a dexamethasone-free, short dosing-period regimen consisting of bortezomib, lenalidomide, and clarithromycin. This regimen was found to be highly effective and succeeded in achieving stringent complete response. CONCLUSIONS: The successful dexamethasone-free regimen clearly shows that dexamethasone is not a requisite component in treating multiple myeloma, and it can be substituted with clarithromycin. This regimen is particularly useful for treating patients with multiple myeloma associated with diabetes mellitus.

Detection of CCND1 Gene Copy Number Variations Using Multiplex Ligation-Dependent Probe Amplification and Fluorescence In Situ Hybridization Methods.

The CCND1 locus is located in 11q13 and encodes the G1-S regulatory protein, cyclin D1. Cyclin D1 is frequently amplified in various types of cancers, and is an attractive potential therapeutic target. Multiplex ligation-dependent probe amplification (MLPA) is a new, high-resolution method for the detection of amplification of numerous genes including CCND1 in small amounts of DNA fragments derived from formalin-fixed, paraffin-embedded material in a single reaction. This approach is, however, based on PCR and averages many different cells, so validation by morphological methods such as fluorescence in situ hybridization (FISH) is theoretically mandatory. Here we describe detection of CCND1 gene copy number variations by commercially available MLPA kits and FISH using a bacterial artificial chromosome (BAC) probe.

Fine needle aspiration cytology findings of myxoinflammatory fibroblastic sarcoma: A case report.

Myxoinflammatory fibroblastic sarcoma (MIFS) is rare low-grade soft-tissue tumor that occurs in extremities. Clinically it is difficult to differentiate from benign lesions, such as nodular fasciitis, and malignant tumors, such as liposarcoma. We report a case of MIFS in the forearm of a 34-year-old man. The resected tumor measured 5.3 × 2.7 × 2.5 cm3 , had a lobular structure with indistinct boundary, and consisted of a large amount of translucent and yellow mucous-like substrate. Cytological examination of a preoperative puncture aspiration specimen showed histiocyte- and fibroblast-like tumor cells in a mucous-like matrix together with scattered lipoblast- and ganglion-like cells. Nuclear chromatin of the tumor cells showed a fine granular appearance but no notable hyperchromasia. There were no clear malignant findings. On Giemsa staining of the tumor cells, there were fine granular and hyaline intracytoplasmic substances that showed purple to reddish-purple metachromaticity. Prominent inflammatory cells were not observed in the specimens. MIFS should be considered in the differential diagnosis for a myxoid tumor in the extremities.

Regulation of p27 by ubiquitin ligases and its pathological significance in human lung carcinomas.

Down-regulation of cyclin-dependent kinase inhibitor protein p27, due to enhanced degradation, is frequently observed in various cancers. The ubiquitin ligases that mediate this degradation have been identified as S-phase kinase-associated protein-2 (Skp2), Kip1 ubiquitylation-promoting complex (KPC), and p53-inducible protein with RING-H2 domain (Pirh2) as well. We investigated the correlation among expression of these 3 ligases and p27 status in surgical specimens of human lung carcinomas by immunohistochemical analysis. Among 93 cases, expressions of p27, Skp2, KPC, and Pirh2 were found in 89.2%, 59.1%, 59.1%, and 67.7%, respectively. Down-regulation of p27 in cancer cells was frequently observed in adenocarcinoma (AC) and squamous cell carcinoma (SCC), but not in small cell carcinoma (SmCC). Overexpression of ubiquitin ligases was variously observed among histological types: Skp2 was more frequently observed in SCC and SmCC, KPC in SCC and Pirh2 in AC, followed by SCC. Several novel findings were obtained: (i) cytoplasmic p27 was observed in 8.6%, most frequently in SCC (13.3%), and correlated with nodal metastasis (P=.0044), (ii) significant inverse correlation between nuclear p27 and Pirh2 expression was observed by statistical analysis and at the cellular level, and (iii) cytoplasmic Pirh2 and total (cytoplasmic and/or nuclear) Pirh2 were significantly correlated with the nodal status (P=.0225, 0.0314), the pathological stage (P=.0213, 0.0475) and recurrence-free survival (P=.0194, 0.0482, respectively) in AC. Altogether, our data suggests that p27 and its cognate ubiquitin ligases are specifically involved in the clinical profiles, and thus, molecular targeting of these ubiquitin ligases, in particular, Pirh2, may have therapeutic value for human lung carcinomas.

Gene amplification of CCNE1, CCND1, and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

New and effective treatments for advanced gastric cancer are urgently needed. Cyclins E and D1 form a complex with cyclin-dependent kinase 2, 4, or 6 to regulate G1-S transition. The G1-S regulatory genes encoding cyclin E (CCNE1), cyclin D1 (CCND1), and CDK6 (CDK6) are frequently amplified in gastric cancer and may therefore influence molecularly targeted therapies against ERBB2 or EGFR when coamplified. A total of 179 formalin-fixed and paraffin-embedded gastric cancer specimens were examined for these gene amplifications by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization. Amplification of at least 1 G1-S regulatory gene was found in 35 tumors (CCNE1 amplification, 15% of samples; CCND1, 6%; CDK6, 1%). In 13 of the 35 tumors, dual-color fluorescence in situ hybridization identified coamplification of the G1-S regulatory genes with ERBB2, EGFR, and/or KRAS in single cancer nuclei. The observation that cells with G1-S regulatory gene amplification contained clonal subpopulations with coamplification of ERBB2, EGFR, or KRAS in 5 early and 3 advanced cancers suggests that amplification of the G1-S regulatory genes represents an early event, which precedes ERBB2, EGFR, or KRAS amplification. Amplified CCNE1, CCND1, and CDK6 in advanced gastric cancer may be potentially useful as direct targets for molecular therapy or for combination therapy with ERBB2 or EGFR inhibitors. Multiplex ligation-dependent probe amplification could be a useful tool for identification of patients who would benefit from such therapies.

MicroRNAs associated with increased AKT gene number in human lung carcinoma.

MicroRNA (miRNA) expression profiles were examined in 3 groups of lung carcinomas that had been stratified by increases in AKT1 or AKT2 gene number. Microarray analysis using 2000 probes revealed 87 miRNAs that were up-regulated and 32 down-regulated miRNAs in carcinomas harboring amplification or high-level polysomy of the AKT1 (AKT1+), as well as 123 up-regulated and 83 down-regulated miRNAs in those of the AKT2 genes (AKT2+), in comparison with carcinomas harboring disomy of both (AKTd/d). In total, 182 miRNAs were up-regulated in AKT1+ or AKT2+, compared with AKTd/d. Among these, 28 miRNAs were up-regulated in both the AKT1+ and AKT2+ groups, with a log2 ratio between 1.02 and 3.71 relative to AKTd/d group, including all miR-200 family members. Quantitative real-time polymerase chain reaction showed that carcinomas exhibiting lymph vessel invasion had significantly lower expression of miR-200a (P=.0230) and miR-200b (P=.0168), regardless of the status of the AKT genes. Moreover, a detailed statistical analysis revealed that, in adenocarcinoma and in the early stage of carcinomas (pathologic stage I/II), expression of miR-200a was higher in the AKT2+ group compared with the AKT1+ group, and these differences were statistically significant (P=.0334 and P=.0239, respectively). However, the expression of miR-200a was not significantly correlated with the expression of its target, the zinc finger E-box-binding homeobox 1 (ZEB1; P=.3801) or E-cadherin (P=.2840), a marker of the epithelial-mesenchymal transition. These results suggest that AKT2 can regulate miR-200a in a histology- or stage-specific manner and that this regulation is independent of subsequent involvement of miR-200a in epithelial-mesenchymal transition.

A new fluorescent anatomic pulmonary segmentectomy using PDD endoscope system and vitamin B2: evaluation in a clinical setting using living animal.

OBJECTIVE: Identification of intersegmental planes is essential for successful anatomic pulmonary segmentectomy. We have previously reported a new fluorescence technique using a PDD endoscope system™ and vitamin B2 for identification of intersegmental planes in ex vivo experiments. In the present study, we investigated and evolved this technique to perform ideal anatomic segmentectomy in a clinical setting using living pig models. METHODS: Cranial segmentectomy in the cranial lobe of the right lung was performed in six pigs using our fluorescence technique. The fluorescent cranial segmentectomy was as follows. After identification of the cranial segmental bronchus, vitamin B2 solution as a fluorescent substance was injected via bronchoscopy. The fluorescent segment was observed using a PDD endoscope system, and the identified intersegmental plane was cut using electric cautery. The operative data collected were the rates of accurate identification of the pulmonary segment and perioperative complications. The duration and light intensity of fluorescence of the target segment were recorded to provide an objective measurement of success. RESULTS: In all procedures, it was possible to identify the target segment by its clear yellow-green fluorescence. The rate of accurate identification of the pulmonary segment was 100%. The fluorescence continued for more than 1 h with adequate light intensity. No perioperative complications were encountered. No unexpected injuries of the major segmental bronchi or vessels occurred. Hemorrhage and air leakage from the transected intersegmental plane were negligible. CONCLUSIONS: Our new fluorescence technique in a clinical setting involving a PDD endoscope system™ vitamin B2 enabled accurate and safe anatomic pulmonary segmentectomy, with enough strong and long fluorescence in living pig lungs.

Overexpression and gene amplification of both ERBB2 and EGFR in an esophageal squamous cell carcinoma revealed by fluorescence in situ hybridization, multiplex ligation-dependent probe amplification and immunohistochemistry.

EGFR and ERBB2 belong to the EGFR gene family. In esophageal squamous cell carcinomas (SCCs), amplification of EGFR or ERBB2 is usually mutually exclusive. EGFR amplification occurs in approximately 15% of SCCs, ERBB2 occurs in less than 5%. Here, we report the co-amplification of EGFR and ERBB2 in an ulcerative and infiltrating-type SCC that measured approximately 4.2 × 2.7 × 1.2 cm with a superficial lesion occurring in the thoracic esophagus of a 72-year-old man. Multiplex ligation-dependent probe amplification using representative tumor sections showed gain of CCND1 and coincident amplification of ERBB2 or EGFR or neither. Immunohistochemistry and fluorescence in situ hybridization revealed that the tumor comprised three cancer-cell populations: well-differentiated SCC with high-level ERBB2 amplification and ERBB2 overexpression, more infiltrative poorly-differentiated SCC with high-level EGFR amplification and EGFR overexpression, and poorly-differentiated SCC lacking any ERBB2 or EGFR abnormality. These three populations each had low-level CCND1 amplification and nuclear cyclin D1 overexpression. This histological topology and gene amplification combinations suggested that genetic instability first produced CCND1 amplification, and then ERBB2 or EGFR gene amplification occurred. It is further speculated that during cancer progression and clonal selection indecisive predominance of either clone caused the rare co-amplification of ERBB2 and EGFR in a single chimeric tumor.

Chitosan tubes can restore the function of resected phrenic nerves.

OBJECTIVES: We previously reported that the phrenic nerve could be morphologically repaired by implantation of a chitosan nanofibre tube (C-tube). In the current study, we investigated whether implantation of C-tubes could improve the function of an injured phrenic nerve using a beagle dog model. METHODS: Seven beagle dogs underwent right thoracotomy under general anaesthesia. An approximately 5 mm length of the right phrenic nerve was resected. Five dogs had a C-tube implantation (C-tube group) and other two dogs did not have the C-tube implantation (control group). Diaphragm movements were longitudinally measured by X-ray fluoroscopy before surgery, immediately after the surgery, and 3, 6 and 12 months after the surgery. The diaphragm movement was determined by diaphragm levels at inspiration and expiration phases, and the excursion difference between them was calculated. At 12 months after the surgery, rethoracotomy was performed to examine electrical phrenic nerve conduction. The C-tube and phrenic nerve were then excised for histological assessment of nerve regeneration. RESULTS: Three of the five animals of the C-tube group showed improvement of diaphragm movement with time. In these three animals, slow phrenic nerve conduction was observed. Histological assessment showed that the injured nerve was connected by newly regenerating nerve fibres surrounded by granulation tissue within the C-tube. On the other hand, the animals in the control group and two animals of the C-tube group showed neither improved diaphragm movement, nor electrical conduction to the diaphragm. No nerve fibre regeneration was found by histology. CONCLUSIONS: Our results suggest that, in addition to morphological improvement, C-tube implantation can functionally improve the injured phrenic nerve by promoting phrenic nerve regeneration.

Diverse involvement of isoforms and gene aberrations of Akt in human lung carcinomas.

Emerging evidence confirms a central role of Akt in cancer. To evaluate the relative contribution of deregulated Akt and their clinicopathological significance in lung carcinomas, overexpression, activation of Akt and AKT gene increases were investigated. Immunohistochemical staining for 108 cases revealed overexpression of total Akt, Akt1, Akt2 and Akt3 in 61.1, 47.2, 40.7 and 23.1%, respectively, and phosphorylated Akt in 42.6% of cases. Expression of total Akt, Akt2 and Akt3 were frequently observed in small cell carcinoma, but phosphorylated Akt and Akt1 were more frequently observed in squamous cell carcinoma. FISH analysis to evaluate gene increases of AKT1-3 revealed amplification of AKT1 in 4.2% and AKT1 increase by polysomy of chromosome 14 in 27.3% of cases. For AKT2, amplification was observed in 3.2% and polysomy of chromosome 19 in 26.3% of cases. AKT3 increase was observed in 40.0% of cases only by polysomy of chromosome 1. Although "FISH-positive" AKT1 and AKT2 gene increases (amplification/high-level polysomy) were found exclusively in the cases overexpressing total Akt, Akt1 or Akt2, respectively, AKT3 increase was irrelevant of Akt3 expression. Statistically, expressions of Akt2, p-Akt and cytoplasmic-p-Akt were correlated with lymph node metastasis (P = 0.0479, P = 0.0371 and P = 0.0310, respectively). Although AKT1 and AKT2 gene increase showed positive correlation with, or trend towards a positive correlation with tumor size (P = 0.0430, P = 0.0590, respectively), AKT3 did not. In conclusion, Akt isoforms are differentially involved in the pathological phenotype of lung carcinoma in a diverse manner. Because abnormality of Akt1/AKT1 and Akt2/AKT2 correlated with clinicopathological profiles, Akt1/2-specific targeting may open a novel therapeutic window for the group showing Akt deregulation.

Expression and regulatory effects on cancer cell behavior of NELL1 and NELL2 in human renal cell carcinoma.

Neural epidermal growth factor-like like (NELL) 1 and 2 constitute a family of multimeric and multimodular extracellular glycoproteins. Although the osteogenic effects of NELL1 and functions of NELL2 in neural development have been reported, their expression and functions in cancer are largely unknown. In this study, we examined expression of NELL1 and NELL2 in renal cell carcinoma (RCC) using clinical specimens and cell lines. We show that, whereas NELL1 and NELL2 proteins are strongly expressed in renal tubules in non-cancerous areas of RCC specimens, their expression is significantly downregulated in cancerous areas. Silencing of NELL1 and NELL2 mRNA expression was also detected in RCC cell lines. Analysis of NELL1/2 promoter methylation status indicated that the CpG islands in the NELL1 and NELL2 genes are hypermethylated in RCC cell lines. NELL1 and NELL2 bind to RCC cells, suggesting that these cells express a receptor for NELL1 and NELL2 that can transduce signals. Furthermore, we found that both NELL1 and NELL2 inhibit RCC cell migration, and NELL1 further inhibits RCC cell adhesion. These results suggest that silencing of NELL gene expression by promoter hypermethylation plays roles in RCC progression by affecting cancer cell behavior.

Semi-comprehensive analysis of gene amplification in gastric cancers using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization.

The prognosis of patients with gastric carcinomas at an advanced stage still remains dismal, and therefore novel therapeutic modalities are urgently needed. Since the successful targeting of amplified ERBB2 with a humanized monoclonal antibody, the amplified genes of other receptor tyrosine kinases such as EGFR, FGFR2, and MET, as well as those of other cell regulator genes, are being considered as candidate targets of molecular therapy. The aim of the present study was to determine the amplification status of 26 genes, which are frequently amplified in solid cancers, in advanced gastric cancers. A total of 93 formalin-fixed and paraffin-embedded advanced gastric cancer tissues were examined by multiple ligation-dependent probe amplification, and 32 cases with 'gain' or 'amplified' status of 16 genes were further examined for the respective gene amplification by fluorescence in situ hybridization (FISH) and for the respective protein overexpression by immunohistochemistry. The frequencies of gene amplifications in advanced gastric cancers were as follows: ERBB2 (13 cases, 14%), FGFR2 (7 cases, 8%), MYC (7 cases, 8%), TOP2A (7 cases, 8%), MET (4 cases, 4%), MDM2 (4 cases, 4%), CCND1 (3 cases, 3%), FGF10 (2 cases, 3%), and EGFR (1 case, 1%). Amplification of the receptor tyrosine kinases genes occurred in a mutually exclusive manner except for one tumor in which ERBB2 and FGFR2 were both amplified but in different cancer cells. Co-amplification of ERBB2 and MYC, and EGFR and CCND1, in single nuclei but on different amplicons, was confirmed in one case each. Attempts at correlating the FISH status with the immunohistochemical staining pattern showed variable results from complete concordance to no correlation. In conclusion, combination of multiple ligation-dependent probe amplification and FISH analysis is a feasible approach for obtaining the semi-comprehensive genetic information that is necessary for personalized molecular targeted therapy.

Thyroidectomy using pure natural orifice transluminal endoscopic surgery in a porcine model.

Surgical procedures for thyroid disease that provide cosmetically acceptable results are in demand. Natural orifice transluminal endoscopic surgery (NOTES) is performed through natural orifices and thus avoids incision of the body wall. This study aimed to develop an incision-free surgical procedure for thyroid lobectomy using pure NOTES with an oral approach. In six pig carcasses, an incision was made between the mandible and subcutaneous tissue under direct vision. After subcutaneous dissection and identification of the hyoid bone, the operative field was developed under endoscopic view. After the thyrohyoid membrane was identified, dissection was continued along the thyroid cartilage until the cricoid cartilage was identified and the thyroid isthmus was reached. An original retractor was inserted between dissected tissues to lift and fix the carcass. The thyroid gland was successfully removed through the incision. Similar macroscopic and histological findings were observed on the normal and treated sides, with no damage to the recurrent laryngeal nerves. The times required for securing the operative field and thyroidectomy improved with each operation. This study suggests the feasibility and safety of using pure NOTES for thyroidectomy through a subcutaneous route with an original retractor.